Abstract for TER20203

Developmental Toxicity Evaluation of 3'-Azido-3'-Deoxythymidine and (-) 2', 3'-Dideoxy-3'-Thiacytidine Co-Administered by Gavage to Swiss Albino (CD-1) Mice on Gestational Days 6 Through 16

Substances:

3'-Azido-3'-Deoxythymidine (CASRN 30516-87-1)

(-) 2', 3'-Dideoxy-3'-Thiacytidine (CASRN 134678-17-4)

Report Date: April 6, 2004

Abstract

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings may not have been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. For more information, see the Explanation of Levels of Evidence for Developmental Toxicity. The findings and conclusions for this study should not be construed to represent the views of NTP or the U.S. Government.

3'-Azido-3'-deoxythymidine and (-) 2', 3'-dideoxy-3'-thiacytidine are nucleoside analogues used in combination for the treatment of human immunodeficiency virus. This study examined potential developmental toxicity of AZT, 3TC and AZT/3TC combinations administered orally to timed-mated mice during embryo/fetal development. Combined treatments were administered at clinically relevant ratios (2:1) up to ~33 times the human therapeutic dose, except that one combination was administered in a ratio of 1:2.5 in order to approximate a treatment previously reported to cause adverse drug interactions in perinatal mice. Higher doses of AZT were not included due to previously reported intrauterine growth retardation in mice.

Total daily doses were equally divided (morning and afternoon) and given at least 6 hours apart. Dose volume was 10 ml/kg body weight at each administration for a total daily dose volume of 20 ml/kg/day. The oral route corresponds to a commonly used route in human patients.

Timed-mated mice were monitored at regular intervals for clinical signs of toxicity, feed consumption, and body weight. At necropsy (gd 17), clinical signs, maternal body weight, liver weight, gravid uterine weight, pregnancy status and number of corpora lutea were recorded. In each gravid uterus, the numbers of prenatal deaths (resorptions and/or dead fetuses) and live fetuses were recorded. Live fetuses were weighed, sexed, and examined for morphological anomalies (external, visceral, and skeletal).

Data were analyzed for differences among all 12 treatment groups and for drug and replicate interactions. Significant replicate interactions (AZT X 3TC X REP or COMBO X REP) resulted in analysis of data within each replicate. A significant global drug interaction (AZT X 3TC) resulted in evaluation of pairwise drug interactions within each AZT/3TC combination group in order to classify the interaction (synergism, potentiation or antagonism). When the overall model was significant, individual drug combinations were compared to their constituent doses. Dose-response trends and differences among groups were analyzed within blocks of treatment groups, specifically (a) control and 3 doses of AZT; (b) control and 4 doses of 3TC; (c) control and 3 combinations of AZT/3TC in the clinical ratio or (d) 0, 200 or 1000 mg 3TC/kg/day combined with 400 mg AZT/kg/ day. When a group-wise comparison was significant, individual groups were compared to the control group (a, b, c above) or 400 AZT group (d above).

One female at 100 AZT/50 3TC was found dead (gd 14). Otherwise, clinical signs were unremarkable. At necropsy (gd 17), 20-24 females per group were confirmed pregnant (87-100% per group). Maternal body weight was unaffected. There was no consistent pattern of effects on maternal body weight change, although some comparisons reached statistical significance. From gd 6 to 9, there was an increasing trend for 3TC at 400 AZT. From gd 12 to 15, 200 AZT/100 3TC and 400 AZT/200 3TC were below their constituent doses of 3TC. From gd 15 to 16, there was a decreasing trend across drug combinations and 200 AZT, 200 AZT/100 3TC and 400 AZT/200 3TC were lower than controls.

Maternal liver weight (absolute) was not affected. Maternal relative liver weight (% body weight) was increased at all doses of AZT, showed an increasing trend across 3TC at 400 AZT, and was elevated at 400 AZT/1000 3TC relative to the constituent dose of 3TC. All of these effects were noted in Replicate I, but only the group-wise comparison for AZT was significant in Replicate II. For both replicates combined, relative maternal liver weight showed increasing trends across AZT and drug combination groups, increases above control at 100 and 400 mg AZT/kg/day and elevation above the constituent dose of 3TC at 400 AZT/1000 3TC. A significant drug interaction was noted at 100 AZT/50 3TC (antagonism), but only in Replicate I. The lack of interaction at higher dose combinations and the absence of a drug interaction in Replicate II suggest that this apparent antagonism between 100 AZT and 50 3TC was a spurious finding.

The number of corpora lutea/dam, number of implantation sites/dam, percent pre-implantation loss/dam, number of live fetuses/dam, and percent male fetuses were not affected. Percent resorbed implantation sites showed an increasing trend across 3TC groups at 400 AZT, and 400 AZT/1000 3TC (7.93% resorbed) was elevated above 400 AZT (2.49% resorbed). For percent litters with one or more resorptions a marginally significant overall drug interaction (p=0.0458) was noted. However, pairwise drug interactions were not significant for individual dose combinations. The 400 AZT/1000 3TC groups were higher than 400 AZT for this parameter. AZT/200 3TC and 400 Otherwise prenatal mortality was not affected.

For fetal body weight, decreasing dose-related trends were associated with exposure to AZT alone and to the drug combinations. For AZT, the low, mid and high-dose groups were 93.8, 93.4 and 82.5% of control weight, respectively (all significantly below controls). Low, mid and high drug combinations were 91.6, 90.1 and 85.6% of control weight, respectively (all significantly below controls). For each drug combination, fetal body weight was below the constituent dose of 3TC.

A spurious trend was noted for percent fetuses with external malformations, apparently due to ascending means across 3TC doses. This was not indicative of a treatment-related effect since 1000 mg 3TC/kg/day (2.90% malformed) was comparable to controls (2.51% malformed). The percent fetuses with visceral malformations showed an increasing trend for 3TC at 0 AZT, and the percent fetuses with skeletal malformations showed increasing trends for AZT alone and for the drug combinations. At 400 AZT/200 3TC, the incidence of fetuses with skeletal malformations was elevated relative to the constituent dose of 3TC. There were no significant effects or interactions for the percent malformed fetuses analyzed for all types combined.

Increasing trends were noted across 3TC groups for the percent fetuses with external variations and percent litters with external variations. Both parameters were elevated at 1000 mg 3TC/kg/day relative to controls. Incidences of hematoma, and clubbed limb or kinked tail (both without bone change) were affected. Increased incidences of skeletal variations were found in the same groups that exhibited reduced fetal body weight. Percent fetuses with skeletal variations showed significant increasing trends for AZT and the drug combinations. Percent fetuses with skeletal variations was elevated above controls at 100, 200 and 400 mg AZT/kg/day, and for 100 AZT/50 3TC, 200 AZT/100 3TC and 400 AZT/200 3TC. Each drug combination, except 100 AZT/ 50 3TC, showed an elevated incidence of skeletal variations relative to the constituent dose of 3TC.

In summary, all treatments were well tolerated by the dams. Dose-related decreases in fetal body weight and increases in skeletal variations were found for all doses of AZT and all drug combinations, but not for 3TC. In the drug combination groups, reduced fetal body weight and increased skeletal variations appeared due to effects of AZT alone, and there was no evidence for adverse drug interactions between AZT and 3TC. The developmental lowest observed adverse effect level for 3TC was 1000 mg/kg/day and the no observed adverse effect level was 200 mg/kg/day. The LOAEL for AZT was 100 mg/kg/day and a NOAEL was not established in this study. The LOAEL for AZT/3TC combinations was 100 AZT/50 3TC and a NOAEL was not established in this study. In conclusion, the combination of AZT with 3TC up to ~33 times the human therapeutic dose did not alter adverse developmental outcome in mice relative to AZT alone.